For the excitation of nonlinear processes (multiphoton absorption, SHG, OBIC, time-resolved fluorescence detection, etc.) by pulsed laser in specimens, the highest possible pulse-peak powers Ppeak are needed. The pulse peak power (intensity I) is calculated as follows:                               P          Peak                =                              P            Avg                                τ            ·            f            ·            A                                              (        1        )            
Accordingly, the shorter the pulse length xcfx84 in the specimen, the higher the peak power. However, short pulses have a determined spectral width xcex94xcex, depending on the pulse length. The repetition rate is designated by A and the interaction surface with the specimen is designated by f. Pavg represents the average power of the laser radiation. Due to dispersion in optical components, the pulses are broadened when traversing the optical media (including the specimen). In addition, nonlinear effects such as SPM can occur, affecting the spectrum and therefore, in turn, the pulse length. There is accordingly a need to optimize the pulse length and the average power at the location of the laser interaction with the specimen.
Short pulses of less than 1 ns can not be directly measured by electronic means because of their brevity. For this purpose, autocorrelators are used, for example; the autocorrelation function of the pulses can be determined in this way. The pulse length is subsequently determined from this autocorrelation function. However, these measuring instruments generally require a parallel beam of the laser light under examination. They are therefore unsuitable for determining the pulse length directly following a high-aperture objective. They are unsuited in principle for determining/optimizing the pulse length as a function of the depth of penetration into a specimen.
It is possible by means of the proposed method to measure the pulse length at the location of laser interaction with the specimen and to optimize the pulse peak power. The nonlinear interaction combined with a linear interaction is used for this purpose in a biological specimen.
In general, the nonlinear interaction can be described as follows:                               S          NL                =                              C            ·                          P              peak              n                                =                      C            ·                                          P                avg                n                                                              (                                      τ                    ·                    f                    ·                    A                                    )                                n                                                                        (        2        )            
where C is a proportionality factor and n is the order of nonlinearity. The constant C depends on the properties of the specimen. Nonlinear specimen interactions are, e.g., two-photon absorption (n=2), the generation of the second harmonic (n=2), three-photon absorption (n=3), etc.
A linear specimen interaction is given, among others, by:
SL=C1xc2x7Pavgxe2x80x83xe2x80x83(3)
where C1 is again a proportionality factor which also depends in this case on the properties of the specimen. A linear interaction is, e.g., any reflection on the specimen, the excitation of a single-photon fluorescence, or the measurement of the average power with a power measuring instrument.
When both processes (linear and nonlinear) are measured at the same time and the ratio                     R        =                              S            NL                                S            L            n                                              (        3.1        )            
is determined between the two signals, the following relationship results, for example, for a two-photon absorption:                                                                         (                2                )                            /                              (                3                )                                      ⁢                          xe2x80x83                        ⁢            R                    =                                                    S                NL                                            S                L                n                                      =                                                            C                                                            C                      1                      2                                        ·                                          f                      2                                        ·                                          A                      2                                                                      ·                                  1                                      τ                    2                                                              =                                                Const                  .                                ·                                  1                                      τ                    2                                                                                      ,                            (        4        )            
where f and A are independent from the pulse length.
The proportionality constants (C, C1) are normally also independent from the pulse length. In the case of two photons, the ratio R of nonlinear to linear signal is inversely proportional to the pulse length and independent from the average power. The constant (Const) depends on the specimen used and on the detection device.
The application of the invention will be explained by way of example with reference to two-photon absorption.